Methods for immunoploidy analysis

ABSTRACT

A method and apparatus are provided for selecting and analyzing a subpopulation of cells or cell objects for a certain parameter such as DNA using image analysis means. The cells are first stained with an alkaline phosphatase technique including a monoclonal antibody specific to a protein in at least one of the cell&#39;s cytoplasm or on a cell membrane, thereby marking any cells including the protein as to type. A second staining of the DNA in the nucleus is accomplished by a Feulgen technique that destroys the cell cytoplasm. After the staining and marking, the cells may then be gated using the image analysis means on the visual parameter such as colored DNA or colored antigen into a subpopulation that is to be measured. The selected cells may then be examined by digital image processing and measured for a parameter such as a true actual measurement of DNA in picograms. A quantitation of the measured parameter may be generated and provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 320,274 filedMar. 7, 1989, now abandoned, which is a division of Ser. No. 121,674filed Nov. 17, 1987, now U.S. Pat. No. 5,016,283, which is acontinuation in part of application U.S. Ser. No. 927,285 filed Nov. 4,1986, in the name of James W. Bacus and entitled "Analysis Method andApparatus for Biological Specimens" and now abandoned, which in turn isa continuation-in-part of a U.S. Ser. No. 794,937 filed Nov. 4, 1985, inthe name of James W. Bacus and entitled "Cell Analysis Apparatus andMethod With Calibration and Control Slide" and now U.S. Pat. No.4,741,843 both of which are commonly assigned with the presentapplication. These previous disclosures of Bacus are hereby expresslyincorporated by reference herein.

FIELD OF THE INVENTION

The invention relates generally to the measurement of cell objectfeatures and other parameters by image analysis, and is moreparticularly directed to quantitative measurement methods and apparatusfor DNA analysis of small cell populations.

BACKGROUND OF THE INVENTION

The present invention is directed to quantitative testing apparatus andmethods which may be used for a wide range of diagnostic and prognosticevaluations of various cells, antigens, or other biological materialstaken from the human body. However, for purposes of illustration andease of understanding, the invention will be disclosed in conjunctionwith its preferred use, which is the quantitative measurement ofcellular DNA for the purpose of cancer diagnosis and prognosis. Morespecifically, the present invention is directed to methods and apparatusfor interactive image analysis which are adapted to analyze and quantifythe DNA in different classes of specimen cells taken from a human body.

The current state of the art in the pathology laboratory for measuringthe DNA content of a cell is by visual observation. A pathologistobserves through a microscope primarily the shape and texture ofsuspected cancer cells and then classifies the cells into a normalcategory or into one of several abnormal or cancer categories. Suchevaluations are very subjective and can not differentiate and preciselyquantify small changes in DNA within individual cells or in very smallpopulations of abnormal cells. These small changes may represent anincipient stage of cancer or a change in cell structure due to treatmentof the cancer by chemotherapy or radiation. Such small changes are,therefore, important in the diagnosis and prognosis of these diseases.

However, the advantage in diagnosis and/or prognosis of abnormal ploidydistributions that a pathologist viewing a specimen under a microscopehas is the discerning expertise of a skilled person in classifying cellsas normal or abnormal. There is an innate human ability to makerelatively quick infinite gradations of classification, i.e., almostnormal, slightly abnormal, etc. On the other hand, the classificationand measurement of cell features and parameters manually by apathologist on a cell-by-cell basis is extremely tedious and timeconsuming. Statistical analysis of such cell data taken by hand isrelatively difficult because each record has to be entered and thenprocessed. For different records, taken at different times, and undervarying conditions broad statistical categorizations may be unreliable.

The alternative is automated cell analysis where the pathologist usesspecialized equipment to perform the analysis. In automatic cellanalysis, such as that accomplished by a flow cytometer, mass tests areperformed in gross on a specimen cell population without a researcherbeing able to exclude or include certain data of the population. Thespecimen is measured "as is" without really knowing what cells are beingmeasured and how many. Important single cell data or data fromrelatively small groups of cells are lost in the overall averaging of aspecimen. Further, relatively large amounts of a specimen have to beused to provide results from these tests and the sample is consumed.

Although there are commercially available general purpose flowcytometers, they are very expensive and can handle only liquid bloodspecimens or tissue disaggregations. These cytometers are incapable ofworking on standard tissue sections or using conventional microscopeslides which are the preferred specimen forms of pathology laboratories.Additionally, a flow cytometer does not allow for the analysis ofmorphological features of cells such as texture, size and shape of cellnuclei and alterations in the nuclear-to-cytoplasmic ratios of cells.

The methods and apparatus illustrated in the referenced Bacusapplications have solved these and other problems relating to theanalysis of various features and parameters of cell objects. Bacusdiscloses a measurement method and apparatus which can acquire accuratequantitative data concerning a plurality of individual cells veryquickly by an interactive process with a pathologist or an operator.

The Bacus apparatus provides means for displaying on a video monitor animage of a group of cells from a field of a microscope slide. The imageis further digitized and stored in a memory of the apparatus. From thedigitized image, a processor means identifies each possible cell objectautomatically by a pattern recognition technique. An interactive programallows the operator to point to each object or cell in succession andmake morphological decisions for classification and measurementsconcerning each. For quantitative DNA analysis, the measurement is ofthe optical density of the cell object and the classification is by apathologist as to whether the cell appears normal or cancerous. Thedecisions include whether to accept or reject a particular cell forfurther measurement and processing. The cell object, if selected, canthen also be classified into one of several classifications for laterstatistical analysis. The apparatus further has means which permit theclassification and storing of more than one image.

When the apparatus is used for DNA analysis, tissue and cell specimensare applied to a slide which is then stained with a specific stain thatcombines proportionately with the DNA and essentially renders invisiblethe remainder of the cell so that the image analysis apparatus canmeasure the optical density of the DNA which is concentrated in thenucleus of the cell. The stain associates with the DNA to provide adetailed nuclear structure and pattern which may be visually observedand interpreted by the pathologist using the apparatus forclassification. The amount of DNA in the malignant cells issubstantially greater than that for normal cells because the malignantcells are usually dividing and replicating rapidly or the malignantcells have abnormal numbers of chromosomes or have defectivechromosomes.

The Bacus apparatus can not only detect minute alterations in thenucleus by providing a real and accurate measurement of the DNA mass inpicograms but also can measure and quantify the amount of DNA and relateit to stored statistical analyses to aid in diagnosis. Morespecifically, the invention allows an iterative analysis of specimenpopulation cells and provides a histogram or other statistical displayof the population distribution of the cells with respect to their DNAcontent and with respect to a standard DNA for normal cells so thatsubtle shifts in population distribution can be readily understood. Tothis end cell nuclei images are not only acquired and stored but thedata therefrom can be integrated with other statistical data to providemultivariable analysis, discrimination of cells, histograms, andscattergrams of cells or cell populations.

While the methods and apparatus described above are extremelyadvantageous and advance the art of aneuploidy analysis by imageprocessing, they are not as sensitive as they could be. With theprogress in measuring the quantity and distribution of DNA in a cellularpopulation, there has come the need to further refine and sensitize thatanalysis and characterization process. One area in which sensitivity ofthe above described process can be improved is in the operatorclassification of cell types.

The previous apparatus of Bacus relies mainly on the pathologist oroperator to make a subjective judgement concerning the classification ofcell types, and whether they are to be classified at all. This is aprincipal advantage of the apparatus where the expertise of thepathologist in discerning cell types is automated and measurement ofspecified parameters of the chosen cells is accurately made. However, ithas been learned that different cell types which are really quitedifferent structurally appear morphologically similar under themicroscope.

This is particularly true when the nuclear DNA has been enhanced byFeulgen staining. Such nuclear staining is for the purpose of enhancingthe optical characteristics of the nuclei of the cells which contain theDNA, but that necessarily de-emphasizes the visual characteristics ofthe cytoplasm in the rest of the cell outside of the nucleus. The resultis to allow easier image analysis and precise measurement of the DNA ofthe nuclear material, but at the same time this enhancement causes theloss of the visual morphological characteristics of the cytoplasm whicha pathologist might use to distinguish one type of cell from another.Additionally, there are different cell types, which it is advantageousto classify separately, but which provide no or only faint visual cluesas to their differences.

Thus, there is the need to alert a pathologist classifying the cellpopulations for DNA analysis with the Bacus instrument about thedifferent cell types, whether by optical enhancement or otherwise. Amore definitive mechanism would be the use of some demonstrable markeron the cells themselves by which the pathologist can objectivelyseparate the various cell types. There are known in the art many opticalenhancement or marking techniques for cell populations, including thosedescribed in the above referenced Bacus applications. For example, sincethe advent of monoclonal antibody production, numerous antibodies havebeen developed which are specific for cellular components, either in thecytoplasm, nucleus or on the cell membrane. Some have already been usedto type cells in pathology to assist in the definition of the cell oforigin of a number of tumors where subjective morphology is equivocal.

Among the most notable of these antibodies are antibodies to LeukocyteCommon Antigens, which identify inflammatory cells, and antibodies to afamily of cytoplasmic structural proteins called cytokeratins whichidentify cells arising from epithelial structures. Other antibodies tocytoplasmic components such as intermediate filaments can be utilized toidentify cells which provide structural support, the so called stromalcells. In addition, numerous antibodies exist which are morespecifically related to individual tumor types.

However, further optical enhancement of the cytoplasm for differenttypes of cells is problematic in view of the current DNA stainingtechnique. There are many difficulties, the least of which is that anoptical enhancement factor for the cytoplasm should be compatible withthe present imaging techniques using computer analysis of opticaldensity and be required to provide such compatibility without impairingthe sensitivity of the imaging techniques for the present nuclearstaining. Chemical compatibility with the present Feulgen stainingtechnique also presents a major hurdle. Optical enhancement of thecytoplasm after Feulgen staining of the DNA is substantially unavailablebecause the Feulgen process is destructive of the cell cytoplasm andchanges the way it appears normally. However, prior optical enhancementof the cytoplasm is equally as difficult because the Feulgen stainingprocess is caustic with the use of highly acidic reagents which caneasily destroy other optical enhancement factors. Moreover, if doneprior to Feulgen staining, the optical enhancement process of thecytoplasm cannot affect the nuclear material in a manner such that theresult of the subsequent Feulgen staining will be changed.

SUMMARY OF THE INVENTION

The invention provides methods and apparatus for the measurement ofselective features and parameters of cells in a population by theoptical identification of their type. More specifically, the inventionmeasures the DNA content of selected cells of a subpopulation which isselected from a larger population based on optically marking certaincells in the population.

In a preferred embodiment the optical marking of the cell types iseffected by binding an optical enhancement factor, such as a chromogen,to a specific protein in the cytoplasm of a cell in order to type acell. Particularly, a monoclonal antibody specific to the cytoplasmicprotein binds to the protein site and is magnified by an enzymedevelopment technique. After certain types of cells in the populationhave been tagged with a protein specific optical enhancement, a Feulgenstaining process is used to stain the nuclear DNA in all of the cells.An imaging apparatus is then used for the computerized image analysis ofthe cell population. The apparatus provides means for displaying on avideo monitor an image of the cell population from a field of amicroscope slide. The image is further digitized and separated into twoseparate images where in the first the DNA stained areas are visible andin the second the optically enhanced cytoplasm areas are visible. Thetwo image areas are combined and those cells which contain opticallyenhanced cytoplasm areas are marked so that the operator can visualizethose specific cells.

From the digitized DNA areas, the imaging apparatus identifies eachpossible cell object automatically by a pattern recognition technique.An interactive program allows a pathologist to point on the videomonitor to each identified object or cell in succession to makedecisions for classification and measurements concerning each. Themarked cells can be specifically excluded from a subpopulation by theclassification process or specifically included. They may further beidentified as to DNA content in a separate classification.

By combining the marking or identification of certain types of cells byan immunohistochemical technique with DNA Feulgen staining, the abilityto perform DNA content analysis with a greater degree of accuracy andsensitivity is enhanced. This greater sensitivity provides at least twomore avenues of diagnostic and prognostic utility for human tumors. Inone method, the immunologic marking can be used to mark which of thecells of a particular population are not derived from the tumor, leavingthe remaining cells which are not marked immunologically to be analyzedfor DNA content. This method is advantageous where a moderate number ofinflammatory cells are present in a tumor. Thus, using an antibody toleukocyte common antigen, the immunological marking can identify theseinflammatory cells so they can be excluded from the DNA assay.Alternatively, when the tumor cells are relatively rare and non-tumorcells make up the majority of the cells available for analysis, usingimmunohistochemical marking which specifically identify tumor cellsprovides a much easier and more sensitive determination of DNA mass fora cell population. In this case, antibodies to cytokeratin are utilizedto identify epithelial derived tumors such as carcinomas. The analysiswill then be focused on these cell types while discarding cells negativefor cytokeratin as being inflammatory or support cells.

One specific embodiment of the invention includes staining of the cellpopulation with an alkaline phosphatase technique utilizing a monoclonalantibody against a specific cytoplasmic antigen. The resulting stain issubstantially specific to the cytoplasm and does not stain the nucleusof the cells. A Feulgen staining process using Thionin is then performedto stain the DNA in the nucleus of each cell. The alkaline phosphatasestaining method is used because of its compatibility with the Feulgenstaining technique. The alkaline phosphatase staining is specific to thecytoplasmic antigen binding the chosen monoclonal antibody and does notharm the nuclear material so that it may receive the Feulgen stain in asubsequent step. The alkaline phosphatase staining is accomplished firstbefore the destruction of the cytoplasm by the Feulgen stainingtechnique. The chromogen chosen for the staining technique is a fast reddye which is advantageous for two reasons. In the first instance thefast red dye which is precipitated is not susceptible to being washedout by the Feulgen stain process and thus will remain for the opticalvisualization. The second reason is that the chromogen providesexcellent optical separation from the blue Thionin dye used in theFeulgen staining process.

Accordingly, a general object of the invention is to provide a new andimproved method and apparatus for analyzing cells or other biologicalmaterials by using image analysis techniques.

Another object of the invention is to provide new and improved methodsand apparatus for making a quantitative ploidy analysis of cells usingimage pattern recognition equipment.

These and other objects, features, and aspects of the invention willbecome apparent upon reading the following detailed description whentaken in conjunction with the appended drawings wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial representation of an image analysis systemconstructed in accordance with the invention;

FIG. 2 is a functional block diagram of the image analysis systemillustrated in FIG. 1 which is adapted to perform the methods for thequantitation of nuclear DNA in accordance with the invention;

FIG. 3 is a schematic block diagram of the image acquisition apparatusillustrated in FIG. 2;

FIG. 4 is a functional system diagram illustrating the major operationsof the system control illustrated in FIG. 2;

FIGS. 5 and 6 are top perspective and cross-sectional views,respectively, of a slide particularly adapted for use in the imageanalysis system illustrated in FIG. 1 and having separate areas forcalibration cell objects and specimen cell objects;

FIG. 7 is a pictorial view at the microscopic level of the bindingeffects of a monoclonal antibody;

FIG. 8 is a graphical representation of the % of light transmission as afunction of light wavelength for the two stains and the two colorfilters used in accordance with the invention;

FIGS. 9, 10, and 11 are pictorial representations of images of a cellpopulation showing an unfiltered image, a red filtered image, and a bluefiltered image, respectively;

FIG. 12 is a functional flow chart of one preferred method ofquantitating DNA for human carcinoma in accordance with the invention;

FIG. 13 is a pictorial representation of the image monitor 37 during theselection process, illustrating the marked cells;

FIG. 14 is a pictorial representation of the many optical fields on theslide illustrated in FIGS. 5 and 6;

FIG. 15. is a pictorial representation of the calibration screen whichappears on the instruction monitor illustrated in FIG. 1;

FIG. 16 is a pictorial representation of the analysis screen whichappears on the instruction monitor illustrated in FIG. 1;

FIG. 17 is a system flow chart of the analysis system screenarchitecture of the image analysis system illustrated in FIG. 1;

FIG. 18 is a functional flow chart of the main menu of the main screenillustrated in FIG. 17;

FIG. 19 is a functional flow chart of the calibrate menu of thecalibrate screen illustrated in FIG. 17;

FIG. 20 is a functional flow chart of the adjust blue boundary menu ofthe adjust blue boundary screen illustrated in FIG. 17;

FIG. 21 is a functional flow chart of the adjust red boundary menu ofthe adjust red boundary screen illustrated in FIG. 17; and

FIG. 22 is a functional flow chart of the analysis menu of the analysisscreen illustrated in FIG. 17;

FIGS. 23A-23D illustrate different histograms for aneuploidy analysis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The apparatus illustrated in FIGS. 1 and 2 and the methods describedherein can be used to develop histograms, and other statistical data, ofcell populations for the diagnosis and prognosis of malignancies andother diseases. Specifically, the quantity and distribution of nuclearDNA in separate or combined classifications of cell populations isavailable. To illustrate the utility of such statistical analysesreference is directed to FIGS. 23A-23D.

Referring now to FIG. 23A there is shown a normal ploidy histogramhaving a typical cell number versus mass distribution for healthy,non-dividing cells. The number of cells is provided on the ordinate axisand their nuclear DNA mass on the abscissa. If the cell population shownin the figure is not dividing, the DNA content should be peaked around anormal peak G0/G1 which is the diploid amount, 7.18 picograms/cell. Thisrelative mass of DNA is labelled as 1.0 to normalize the abscissa of thehistogram. FIG. 23B also shows a normal cell population which isdividing, such that there is a significant G0/G1 peak at 1.0 and asecond peak G2 at 2.0. The peak at 2.0 is normal because some of thecells are in division and have double the normal diploid amount of DNA.The saddle S between the two peaks is relatively low and does notindicate any malignancy.

Comparing the histogram in FIG. 23C with the first two, it is seen thatthis cell population is skewed from normal by having a higher first peakaround 1.5 and second peak around 3.0. Further, the saddle S is morepronounced and can be rough in cell count. This histogram may show amalignancy because of the abnormally high DNA content for many of thecells. This high DNA content is likely indicative of the increasedploidy amount of malignant cells which are rapidly dividing.

Likewise, in FIG. 23D it is shown that the G0/G1 peak occurs at 1.0 witha normal diploid amount of DNA but has a relatively large trailingsaddle from 1.0 to 2.8. A normal G2 second peak is not noted and isindicative of an abnormal cell population. The shape of the histogram islikely due to abnormal DNA amounts in cells and clones of cellsindicative of malignancy. Therefore, from the shapes and changes in celldistribution histograms, diagnostic and prognostic information can beobtained.

DNA analysis of human cells has been shown to have both diagnostic andprognostic utility for human tumors. As with any test, its usefulness isdependent on both the accuracy and sensitivity of the technique employedfor the analysis. If a tumor specimen were composed only of the tumorcells, the accuracy and sensitivity of the illustrated technique wouldbe a function of the DNA staining and the accuracy of the measuringinstrument. However, tumors are most commonly composed of a mixture ofcell types. In addition to the tumor cells one finds the normal tissuefrom which the tumor arose, supportive and structural elements and avariety of inflammatory cells and cells which are part of the repair anddefense process of the host. These cells vary in amount from tumor totumor and may indeed numerically overshadow the tumor cells in manycases.

If non-tumor cells are included in the histograms of the DNA analysisillustrated, several errors can occur:

1. An insufficient number of tumor cells may be identified resulting ina tumor inappropriately being assigned a normal DNA content;

2. In tumors with a normal DNA content, the normal cells will exaggeratethe peak on the histogram where the resting tumor cells appear andartifactually lower the percentages of proliferating tumor cells;

3. If the non-tumor cells themselves are proliferating, they will givean artifactual elevation to the assessment of proliferating activity inthe tumor.

Thus, an improvement to the DNA analysis could be made with a mechanismto appropriately eliminate irrelevant cells. Among the potentialmechanisms are to attempt to distinguish tumor cells from non-tumorcells by cell size and shape characteristics, either quantitatively orby subjective morphologic assessment by a pathologist. The quantitativemethod is not useful in that tumor cells themselves can varysignificantly in size and shape and there is substantial overlap betweenthese parameters in tumor cells and those seen in the non-tumor typecells. The subjective morphologic method is more useful in that it takesinto account multiple diagnostic criteria. The present apparatus takesadvantage of this by allowing the pathologist to use his subjectiveskills to separate the tumor cells from the non-tumor cells for DNAanalysis. One problem is that the pathologist traditionally usescharacteristics of both the nucleus and the cytoplasm to make thesesubjective judgments. However, when dealing with the previouslydisclosed method of analysis, only the nucleus is stained making anymorphologic assessment more difficult. The invention solves this problemby the optical enhancement or the marking of selected cells, whichexhibit a certain characteristic or type to identify them immediately.

In the implementation shown, the system is a computerized image analysissystem designed to measure a number of cell object features andparameters from their image on a typical glass slide. The instrumentincludes a sophisticated digital image processing system controlled bysoftware to perform quantitative analysis on individual cells fornuclear DNA content by Feulgen staining as well as measurement of othernuclear features. The imaging system couples the ability of apathologist to identify and classify cells to be studied with thecapability of computer enhanced, high resolution digital video imageprocessing to quantify optical and stain density accurately. Further,the system optically marks certain types of cells such that thepathologist in making his classifications can include or exclude themfrom the study to improve the sensitivity of the process.

In general, a pathologist first prepares a needle aspirate preparationof fresh tissue. The sample is first stained with a alkaline phosphatasetechnique using a monoclonal antibody specific against an antigen in thecellular cytoplasm. The nuclear DNA in the sample is then stained by theFeulgen technique using Thionin as the dye or optical enhancementfactor. After fixation and staining, the preparation is ready foranalysis.

The operator has the option of classifying the cells morphologicallyinto any one of six categories or rejecting inappropriate cells ordebris. The cell data are processed by a system control and the cellularelements are characterized by a quantitative DNA analysis for each cellclass. The information when compared with either a standard cellcalibration or published data allows a pathologist to accuratelyquantify and classify abnormalities that might ordinarily be describedonly verbally from the image.

The addition of quantitative data enables pathologists to perform theirwork in a more standardized and reproducible manner. The system is ofvalue in classifying lesions that may be malignant and in providingprognostic information for known malignancies based on DNA content. Theimage identification system is more advantageous than common flowcytometry methods of evaluating DNA content. Flow cytometry permits anoperator to classify neoplastic cells based only on cell markers. Thepathologist, however, never sees the cells that the instrument hasexamined. In addition, the cell preparation must be used in a short timeframe and is consumed in the course of the study. Although a permanentsection of a tumor under study may be examined at the same time, thereis no guarantee that the same cells are examined in both areas. Also thequantity of tumor available may not be large enough to permit a flowcytometric examination.

In the invention, the quantitative DNA analysis is performed rapidly forthe measurement of DNA and ploidy distribution pattern in a cellpopulation under study. The pathologist advantageously selects the cellswhich are to be used in the population measurements. The measurement ofDNA content is useful and believed to be relevant in diagnosing anddetermining prognosis for a variety of tumors that involve the breast,colorectum, and prostate. The system takes advantage of the skill of thepathologist and the selected cell marking to visually identify andclassify abnormal cells, and then uses the computer aided imaginganalysis to analyze quantitatively those particular cells selected forthe parameters desired. Such instrument advantageously extends andaugments the recognition and diagnostic skills of the pathologist.

With reference to FIGS. 1 and 2 of the drawings, the invention isembodied as an apparatus 11 (FIG. 1) which functionally operates as adigital image analysis and processing system 13 (FIG. 2). The apparatus11 comprises a high resolution microscope 15 with which an operator canview magnified specimens on a support which, in a preferred embodimentis a glass slide 14. The microscope 15 includes adjustment orpositioning means 70 for focusing its optics 16 on the slide 14 and aplatform 51 movable incrementally in X and Y directions via positioningmeans 12 and 17 in order to view different areas thereof. Positioningmeans 12, 17 and 70 are the form of mechanical adjustment verniers whichare conventional for instrument quality microscopes.

The specimens in the field under study are further viewable by theimaging system 13 via image acquisition apparatus 18 (FIG. 2). Theapparatus 18 receives the light intensities of the image of the fieldand converts them into two analog signals (Red, Blue) which can besampled and processed by the image analysis system 13. The imageanalysis system 13 is controlled by a system control 22 in the form of adigital processor such as a personal computer.

An operator, such as a pathologist or laboratory technician, caninteractively communicate with the system control 22 via a keyboard 36,and interacts further with the system to quantitate nuclear DNA andclassify cell objects by the viewing of two displays or monitors. Afirst display, image monitor 37, is a conventional RGB video monitorwhich displays through the system control 22 and the image acquisitionapparatus 18, the same image field as seen through the microscope 15. Asecond display, instruction monitor 62, is another conventional RGBvideo monitor and is used to provide the operator with interactiveprompts, messages, information, and instruction screens from a systemprogram executed by the system control 22.

The keyboard 36 is preferably a conventional AT type keyboard which hason the left-hand side a plurality of function keys F1-F10, in the middlea plurality of alphanumeric keys including the special keys of ENTER,SHIFT, CONTROL, and ALTERNATE, and on the right-hand side cursor controlkeys including up, down, left and right arrow keys, a numeric keypad, anumeric lock key, and an escape key. A keyboard interface 35 translatesthe keystrokes of the operator into numerical codes recognized by thesystem control 22 for specific key indications. A printer 38 is providedfor producing reliable hard copy output of the statistical data andreports produced by the apparatus 11.

A functional schematic of the apparatus 11 is illustrated in FIG. 2 asimage analysis and processing system 13. The image processing system 13is used to analyze a plurality of specimen cell objects on the supportor glass slide 14 of the microscope 15. Suitable high resolutionmicroscope optics 16 receive light from a variable intensity source 19and transmit the light through the slide 14.

Because the source 19 transmits light through the cell objects on slide14, the optical density of each pixel of the image will convert thelight into a different intensity depending upon its percentage oftransmission. Areas with no cell objects in them will appear relativelylight or intense and areas having nontransmissive objects will appeardarker. In general, unmodified cell objects are relatively transparentand their features difficult to distinguish. Staining the cell objectsallows an optical enhancement of the features stained so they willappear darker than other features and their background.

The optical image of each of the cell objects on the slide 14 passesthrough an optical image splitter 25. On one side of the splitter 25,the image acquisition apparatus 18, or other detector, converts theoptical images point by point into two scanned electronic signals (Red,Blue) representing a monochromatic representation of the opticalintensity of each point in the image on the other side of the splitter25, a true color image of the field is provided to the operator byviewing optics 24.

FIG. 3 illustrates the optical filtering and splitting of the imageperformed by the image acquisition apparatus 18. The focused imageformed by the light intensities is transmitted substantially verticallythrough the slide 14 (not shown) and enters the beam splitter 25 mountedin a holder 53. The first true color image passes verticallytherethrough. A second true color image is further transmitted by thebeam splitter 25 perpendicular to the vertical path through the focusinglens 154 to image acquisition apparatus 18. The image acquisitionapparatus 18 comprises a plurality of optical elements including asecond image splitter 156, mirrors 158, 160 and 162, and twomonochromatic optical filters 164 and 166. The image acquisitionapparatus 18 further includes dual video cameras 168 and 170 which eachreceive a portion of the split image. After the second true color imageis split from the microscope optics, it passes into the second beamsplitter 156 where along one path the image is reflected by mirror 158through filter 164 and imaged by camera 168. Along a second path, theimage is reflected from mirror 160, to mirror 162, and then through asecond filter 166 to be imaged by camera 170. The filters 164 and 166are narrow bandpass filters substantially blocking all light frequenciesoutside their pass band. The images from cameras 168 and 170 aretherefor essentially monochromatic images of the field under study onthe slide 14. The first filter element 164 comprises a red filter whichpasses on light of a narrow bandwidth of wavelengths near 620±10nanometers. The second filter element 156 is a blue filter which passeslight of a narrow bandwidth of wavelengths near 480±10 nanometers.

Each television camera 168, 170 converts the monochromatic opticalimages point by point into a scanned electronic signal representing theoptical intensity of points in the image. The output of the cameras 168and 170 which is formatted as a standard NTSC analog video signal isapplied to an analog to digital converter of a pair of image processinginterfaces 21, 23 as seen in FIG. 2. Each image processing interface 21,23 samples the analog signal from each camera 168, 170, respectively,and converts the image signal to a digitized signal which is receivedand stored by the system control 22. Because of the continuous scanning,a real time image of the area that the optics 16 are focused on isprovided by the image display 37. With the dual camera arrangementeither a red color image or blue color image is available simultaneouslyto the system control 22. In general, each monochromatic digital imageis stored as a 512×512 array of pixels where each pixel has a measuredlight intensity of 0-255 (8 bits).

Because the viewing optics 24 of the microscope 15 are located on theother side of the image splitter 25, this parfocal arrangement allowsthe same image seen in the viewing optics 24 to be displayed on theimage display 37. This feature allows the positioning of the platform 51by the manual X, Y adjustment of positioning means 12 and 17 until theoperator views a field of interest on the slide 14. At that time, thecomputer enhanced digitized image of the selected field is displayed onthe image display 37 for further analysis. An X position sensor 26 and aY position sensor 27 generate position signals to a position interface34 which digitizes these signals to provide the apparatus 11 with anaccurate coordinate representation of the field in view.

Both of the displays 37 and 62 are controlled by the system control 22through standard video monitor interface circuitry 39 and 61,respectively. Similarly, the keyboard 36 and the printer 38 communicatewith the system control 22 through conventional interface circuits 35and 41, respectively In addition, the system control 22 controls arandom access memory 73 and other bulk memory storage in the form ofeither floppy and hard disk drives 75 through a memory control interface71.

All of the interface circuits 21, 23, 34, 35, 39, 41, 61, and 71 can beselectively embodied on printed circuit boards which are mounted in thebackplane or card connector of a conventional personal computer formingthe system control 22. Preferably, the personal computer can be onemanufactured by the IBM Corporation having a model designation AT, orthose compatible therewith. Such system control 22 can be run under adisk operating system such as PC DOS, version 3.1 or later. The systemsoftware for the image analysis is called as an application program fromthe disk drive 75, and could for example, be supplied on a floppy disk77. The system software is read from disk 77 and loaded into RAM 73.After loading, program control is transferred to the system softwarefrom the operating system to regulate the various hardware elements ofapparatus 11 previously set forth in a known manner.

The image analysis system 13 operates under an interactive programcontrol by providing a number of instruction screens or images on theinstruction monitor 62 to assist the operator in the quantitation ofnuclear DNA found in one or several cell subpopulations displayed onimage monitor 37. Through interactive responses by the operator and menuselections on different instruction screens, the basic system functionsof the image analysis are performed.

The system functions are more fully illustrated in FIG. 4 where softwarecontrol logic functions for the hardware in block 80 are showncommunicating with software analysis and measuring functions of thesystem software in blocks 82-96. Software is included in the system toperform an initialization and an interfacing of the operating systemfunctions and overall control of the apparatus 11 by instrument controllogic. A screen handler for the instruction screens and the videodisplay of the digital images of the specimen is performed for both ofthe monitors 37 and 62 by image and instruction monitor control logic.The memory and disk storage functions are handled in the software bymemory control logic. Input and output for the interactive responses andreports are handled by the printer and keyboard control logic. Further,data from the cameras 168, 170 and from the position sensors 26, 27 arehandled by image acquisition control logic and position acquisitioncontrol logic, respectively.

The control logic of the software forms an operating shell which is usedby the analysis and measuring functions in blocks 82-96 to control thehardware of apparatus 11 to perform the particular function needed. Thesystem provides a patient labeling function 82 to identify theparticular tissue samples which are under study. Light calibration andposition calibration functions 84 and 86, respectively, are used todetermine a correct reference optical density for a particular field andthe location of that particular field with respect to a coordinateorigin. A control cell calibration function 88 provides a datum for thecompensation of different background stainings and DNA indexcalibration. A boundary formation function 90 allows the operator tochoose a reference level against which the grey scale values of an imageare compared for either the red image or the blue image. A selected cellmarking function 91 provides for the marking of those cells identifiedby the cytoplasm optical enhancement in the acquired data function. Thecell data acquisition function 92 provides for the storage of the greyscale values of the measurements of a specimen image. A cellclassification function 93 allows the operator to classify the acquiredcells, taking into account those marked cells, into differentcategories, and a cell analysis function provides different statisticalanalyses of the categorized data. A utilities function 94 provides theneeded auxiliary type programs for assisting in the primary functions ofthe image analysis. A report generation function 96 is used for hardcopyproduction of analyzed and compiled data from the system on the printer38.

The support on which a specimen is viewed preferably is a transparentglass slide 14 as illustrated in FIGS. 5 and 6. Glass slides of arectangular shape come in standardized sizes such as 1" by 3" and suchcan be used with the following modifications. The slide 14 ispartitioned into two sections where in a first control section 56 arelocated control cell objects 40. In a second section, specimen section58, there are located specimen cell objects 52 which are to be measuredfor their content of DNA. The slide 14 further includes a border 54around the control section 56 for rapid identification of that section.Further, on some convenient location of the slide 14 is placed aidentifying mark 53. The mark 53, illustrated as a cross in FIG. 4, isused as a landmark for identifying the coordinate origin for fields onthe slide.

Because the apparatus 11 may be used in various offices such aspathology offices having persons of varying degrees of skill andknowledge about image analysis, the microscope light source 17 may bevariously adjusted by different operators such that the background mayhave a different light intensity not only from machine to machine butalso at different times depending on the age and nature of the lampdoing the illumination. When the cell objects are DNA nucleus, thestained nuclei appear darker and have high darker gray levels than thecells which have fewer or no DNA content. The particular light intensitylevel is desired to be known in an accurate and real manner; and hence,it is important that there be a calibration of the light intensity toeliminate errors which might be introduced if differences in lightintensity levels are not accounted for.

A further problem with widespread usage of equipment of the foregoingkind is the Feulgen staining factor by which is meant that the user maybe applying either a heavy amount or a light amount of the Thioninstain. This will result in a variation of the gray level being viewedthrough microscope 15 and by the cameras 168, 170 which is then analyzedas to the particular DNA content. Thus, there is a need that theapparatus 11 be calibrated to eliminate differences because of thestaining factor so as to provide a true indication of the actual amountof DNA being analyzed.

In accordance with the present invention, a calibration material 40 isprovided on the slide 14 which, when viewed by the operator under acalibration step of the system software, allows the operator to adjustand to calibrate the apparatus prior to the measuring and analyzing ofspecimen cell objects on the slide 14.

In the illustrated embodiment of the invention there are provided twodifferent materials on the slide 14 with the first being the controlcell objects 40 which are stained simultaneously with the staining ofthe specimen cell objects 12 The simultaneous staining permits theanalysis of the control cell objects to be compared to a predeterminedstored reference light intensity, gray level, or optical density whichthe control cell objects 40 have after staining. If the cell objects arestained either too lightly or too heavily, the amount of understainingor overstaining can be quantitatively analyzed and adjusted for as willbe described hereinafter.

The control cell objects 40 are, in this illustrated embodiment of theinvention, rat liver cells of a known size and shape, and DNA content.The control cell objects 40 may be other types of cells having darkcenters or nuclei which stain well, such as chicken blood cells or troutcells. On the other hand, the cell objects 40 may be artifacts printedon the slide to have a cell shape. Furthermore, as above explained, thecell objects 40 may be conventional plastic beads of a predeterminedsize which will react with a particular fluorescent stain or enzymestain when treated simultaneously with specimen cell objects such asmonoclonal antibodies used in the specimen area 58 of the slide. Thereference cell objects will vary from test to test and the presentinvention is not limited to any particular test or cell objects.

A pathologist will take a slide such as shown in FIGS. 5 and 6 havingpremounted thereon the control cell objects 40, and add thereto thespecimen cell objects 52 which are, in this instance, cells from aneedle aspirate of tumor tissue or monolayer of blood cells or othercells, at the area 58 on the slide. The pathologist will then stain orotherwise treat simultaneously the control cell objects 40 and thespecimen cell objects 52 for image enhancement.

A kit is provided with the apparatus 11 which contains the slide 14 withthe control cell objects 40 thereon, and bottles of reagents which areneeded for the dual staining technique. For the alkaline phosphatasestaining technique the kit contains bottles of a primary antibodyreagent, a biotinylated secondary antibody reagent, an Avidin-Biotin,alkaline phosphatase reagent, and a chromogen substrate (preferably fastred). For the Feulgen staining technique the kit contains bottles ofThionin reagent solution and rinse reagent.

To prepare a slide 14 for analysis, the following process is used. Theslide 14 having control cells in section 54 and specimen cells insection 58 is first stained with the alkaline phosphatase technique tooptically enhance a specific cytoplasmic antigen. Theimmunohistochemical staining begins with a nonfixed specimen on slide 14which is initially cold fixed at 40° C. in acetone for 20 minutes. Theslide is then rinsed twice in a phosphate buffered saline solution forfive minutes (each rinse) without allowing the slide to dry. The slide14 is then incubated for 15 minutes at 37° C. in a moist environmentwith a solution of 2 ml. to which has been added 10 drops of normalhorse serum. This step prevents much of the nonspecific binding of theantibody to sites of the cell objects.

After draining the excess of the normal horse serum solution off theslide 14, it is incubated 15 minutes at 37° C. in a moist environmentwith the primary antibody which binds to the antigen in the cytoplasm ofthe cell objects.

The slide 14 is again twice rinsed in a phosphate buffered salinesolution for 3 minutes (each rinse) without allowing the slide to dry.Next, the slide is incubated for 15 minutes at 37° C. in a moistenvironment with biotinylated bridging antibody solution. The dilutionof the antibody solution being 1:400. The slide is again twice rinsed ina phosphate buffer saline solution for 3 minutes (each rinse).

Thereafter, for development and magnification an Avidin-Biotin, alkalinephosphatase solution is incubated with the slide 14 for 15 minutes at37° C. in a moist environment. Solutions of A-B complex and alkalinephosphatase solution are available from the Vector Corporation ofBurlingame, Calif., as solution A, and solution B of kit number SK-5100.50 microliters of solution A and 50 microliters of solution B is mixedwith 5 milliliters of a 1% Bovine serum albumin/phosphate bufferedsaline solution to form the development solution.

The slide is again rinsed twice in a phosphate buffered saline solutionfor 3 minutes (each rinse). A chromogen substrate is then added to colorthe developed precipitate. Preferably, the substrate is red dye, fastred, from the same kit as above which contains dye solutions 1, 2, and3. Two drops each of solutions 1, 2, and 3 are added to 5 milliliters of100 mM TnS with a pH of 8.2. This solution is incubated with the slidefor 15 minutes at 37° C. in a moist environment. The final step in thecytoplasm development is to rinse the slide for 1 minute in distilledwater.

The slide 14 is then stained with the Feulgen technique using Thionin tooptically enhance the nuclear DNA of each cell. The slide 14 is fixed in10% by volume buffered formalin, adjusted to a pH in the range of fromabout 7.2 to about 7.5, for 10 minutes at room temperature. The nuclearDNA of the cell objects is then hydrolyzed by treating the slide 14 forabout 60 to 75 minutes in 5N hydrochloric acid. The staining process isaccomplished on the slide by transferring it to a Thionin solution forabout a one hour period. Afterward, the slide 14 is washed in a threestate process of rinse solution. The slide is placed in a first stage ofrinse solution for about 30 seconds, transferred to a second stage ofrinse solution for about 5 minutes, and then permitted to stand in athird stage of rinse solution for about 10 minutes. The slide issubsequently washed for about 5 minutes in running distilled water, andthereafter washed with acid alcohol (0.37% hydrochloric acid, 70%ethanol) for 5 minutes. The slide 14 is then dehydrated in absoluteethanol for about 5 minutes to prepare it for coverslipping. Finally,the slide is cleared in xylene for about 5 minutes before being mountedwith a synthetic resin and coverslip.

In FIG. 7 there is shown a representative drawing of the marking andamplification of a particular antigen site 180 labelled AR. The site isan antigenic against a primary antibody 182 that binds thereto. In thepreferred embodiment, a bridging antibody 184 against the primaryantibody is used to bind to the primary antibody and has affixed aBiotin molecule 188. To the bound primary and bridging antibodies isadded Avidin-Biotin complex including an Avidin molecule 186 and threeBiotin molecules 188. These Biotin molecules 188 are conjugated withmolecules of alkaline phosphatase AP enzyme 190. The fourth Biotinmolecule site is open to binding the complex to the bridging antibody184. The site, when a dye such as fast red molecules 192 in solution isadded to this mixture, the alkaline phosphatase reacts with the dyemolecules to produce insoluble fast red molecules 194 which mark theantigen site. While the Avidin-Biotin complex is exemplary andpreferred, any number of different marking techniques can be used.Alternatively, a bridging or primary antibody which is anti-alkalinephosphatase can be used, and then amplified by fast red dye in themanner previously described.

A dual filtering method is thereafter applied to distinguish the areas(cytoplasm) stained by the red chromogen and the areas (DNA) stained bythe blue Thionin. These images, one by the red filter and the other bythe blue filter, are used to separate the DNA stained areas from thecytoplasm areas containing the specific antigen, and to separate both ofthese areas from other cell or field features.

The results and desirability of this dual filtering of a stained cellimage are more fully illustrated in FIG. 8. The percentage of lighttransmitted through the nuclei stained with Thionin dye is shown in thecurve A as a function of the wavelength of light. The percentage oftransmission of light for fast red dye is shown in curve B as a functionof the wavelength of light. The bandwidth of wavelengths of light passedby the blue filter is illustrated in band C while the bandwidth ofwavelengths of light passed by the red filter is illustrated in band D.

When a true color image of a cell population or specimen is filteredwith the blue filter 166, substantially all of the areas stained withthe fast red dye will be invisible and substantially all the areasstained with Thionin dye visible. This is because the Thionin curve Ahas a relatively nontransmissive peak near this wavelength band (480nms.) while the fast red curve B is relatively transmissive in thisband. Thus, in this manner the areas with Feulgen stain can be separatedfrom the cytoplasmic areas. At the other extreme of the graph, the bandD of the red filter 164 is positioned at a place where just the oppositeoccurs. The Thionin curve A is relatively transmissive in this bandwidthwhile the fast red curve B is relatively nontransmissive. Thus, thecytoplasmic areas containing the fast red dye can be identified withouta problem.

Because of the opposite relative differences in light transmissionbetween the two stains in the two filtered bandwidths, the Thioninstained area is enhanced during one filtering relative to other areas ofthe cell, and the fast red stained areas are enhanced relative to otherareas of the cell during the second filtering. While the implementationshows a convenient and advantageous method for discriminating betweenthe two areas having separate staining, it is recognized that there arevarious other staining or optical enhancement methods and filteringmethods which can be used to optically enhance one particular area orfeature over another cell area.

The system software for DNA analysis can now determine the mass of thecellular DNA by obtaining the optical density of the specimen cells fromthe Thionin stain via the instrument 11. In general, the mass of the DNAof a stained cell object can be obtained from its optical density byutilizing the Beer-Lambert Law which is well known in the art ofmicrospectrophotometry. The equation states: ##EQU1## where M=mass ofthe object in picograms

α=spot size in μm²

Eλ=extinction coefficient of the stain at wavelength λ in μm² /pg.

OD=optical density of each spot (dimensionless)

The instrument uses this law to find the mass distribution of a numberof cells or cell objects which can then be analyzed according to astatistical basis, histogram, or other analytical format as will bediscussed hereinafter. The spot size α is determined by the number ofpixels which are measured by the camera 18. The optical density for eachpixel is calibrated by adjusting the light level, focus, and reading areference optical density from the calibration area on the slide. Thiscalibration allows the conversion of the measured light levels for eachpixel into an optical density, a dimensionless quantity.

A calibration for the extinction coefficient is accomplished bymeasuring the optical density for a plurality of the control cells 40 todetermine a peak for the distribution in relative mass units. Becausethe peak DNA content is known for the control cell distribution, thecells in the measurement field can be measured using the relative ODunits and then converted directly into picograms by using the controlcell calibration. For example, if the control cells are known to containpg of DNA (rat liver cells) and a group of calibration cells show a peakdistribution of 11,000 relative OD units then a normal group of humancells (with a known DNA content of 7.18 pg.) would exhibit a peak intheir distribution at approximately 13,250 relative OD units. Further,any other relative OD unit measurement can be converted directly intopicograms by determining and using the extinction coefficient found fromthe group of calibration cells.

The method of the invention is combined with this technique as will nowbe more fully described with respect to FIGS. 9-12. FIGS. 9-11 arepictorial representations of the true color image of a field of a slide14 (FIG. 9), an image filtered with the red filter (FIG. 10), and animage filtered with the blue filter (FIG. 11). FIG. 12 is a flow chartof the steps in the method to produce quantitation of nuclear DNA.

In FIG. 9 there is illustrated several cells of a subpopulation from oneof the fields of the microscope slide 14. The subpopulation containsdifferent types of cells, wherein specific cells 202, 204 have beenoptically enhanced by the alkaline phosphatase staining. All the cells200, 202, 204, 206, and 210 have had the DNA in their nuclei opticallyenhanced by Feulgen staining with Thionin dye.

When filtered with the red filter in FIG. 10, only those areas whichcontain the fast red dye are visible. These are the cytoplasmic areas212, 214 of cells 202, 204 respectively which have been opticallyenhanced because they contain a specific antigen which binds themonoclonal antibody of the alkaline phosphatase staining. These type ofcells 202, 204 are different from the cell types 200, 206, 208, and 210,which are invisible in this image. Further, the nuclei of all cells 200,202, 204, 206, 208, and 210 can be made invisible in the backgroundbecause of the optical separation of the Thionin dye and fast red dye.

In FIG. 11 there is illustrated the result from the blue filter, whereall the nuclei 216, 218, 220, 222, 224, and 226 from the cell populationare visible. The image filtered with the blue filter produces anexclusion of those cytoplasmic areas which are not nuclear stained(clear rather than stained), and which are optically different (fast redstained) even if stained.

The areas stained above the thresholds set for each filtered image canthen be combined by overlaying the DNA image on the cytoplasmic imagedigitally to present on the monitor a clear image of the DNA nuclearareas for typing and analysis where certain cells 202, 204 are markedclearly as to type by an identifying cytoplasmic ring or crescent on thenucleus in FIG. 13. The DNA analysis then proceeds by the interactiveclassification of each cell in the image displayed on the image monitor37. The specifically marked cells 202, 204 can be included in any class,excluded from any class, or classified entirely separately from anyother class. Further, it is evident that different optical enhancementsand filterings will give rise to different typings and increase thesensitivity of the classification process.

The method of measuring and analysis of nuclear DNA using the markingtechnique of the invention is more fully illustrated in FIG. 12. In afirst step, in block 250, a slide 14 containing control cell objects andspecimen cell objects is stained with the alkaline phosphatase techniqueusing fast red dye. The monoclonal antibody is specific against acytoplasmic antigen, for example, Leukocyte Common Antigens orCytokeratins. The next step in the process is to stain the slide 14 withthe Feulgen process using Thionin in block 252. After mounting, theslide 14 is placed on the platform 51 of the instrument 11 and theoperator positions the slide 14 such that a clear field is shown on theimage monitor. The light level is then set for the instrument in block254.

The platform is then moved to the control cell area where an image of asubpopulation of the control cells appears on the monitor 37 in block256. This image is the filtered image (red) showing only the Feuglenstaining. The amount of staining to determine the DNA index, such thatmass can be determined from optical density, is found by measuringoptical density of the control cells in block 260. Normally, more thanone field of control cells is measured to obtain an accurate measurementand this step can be repeated by looping through block 264 and block262. In block 262, the operator moves the platform 51 so that anotherfield of control cells comes into view.

The measurement of the peak of the optical density units is convertedinto the DNA index and stored. The instrument may now be used to measureand analyze the DNA of specific cells on the specimen section 58 of theslide 14. To this end, the instrument platform 51 is moved to a fieldwhere specimen cell objects are visible.

Initially, a cytoplasmic image of the specimen field is obtained usingthe blue filter in block 266 and its boundary set in block 268.Thereafter, a DNA image of the specimen field is obtained using the redfilter in block 270 and its boundary set in block 272. These filteredimages are real time images of the field and are being constantlyupdated by the image acquisition means 18 of the system 11. Theapparatus 11 combines the two filtered images in block 274 to mark theselected cells on the image monitor 37 while displaying the DNA nucleararea. The program then proceeds to a classification step in block 276.When in the classification mode the image acquisition and combination(marking) halts and a static image is presented on the image monitor.

The cells in the image on monitor 37 are then classified as to type byan interactive process with an operator where each cell is pointed to bythe apparatus, and the operator in response to the identificationselects a classification for it using the nuclear morphology and thecytoplasmic markings of the combined image. The classified cells arethen measured for DNA content in block 278 and the results of themeasurements displayed in block 280. The display can be in various formsand statistical analyses of the different classifications orcombinations of classifications.

The measurement step can include more than the cells in one field bylooping through block 282 and block 284. The operator moves the platform51 of the apparatus 11 to another specimen field in block 284 and themarking and imaging steps proceed as previously described. The dataaccumulated in the measuring step for the new cell population is addedto that developed for the previous cell population(s). The display stepin block 280 can be delayed until a significant amount of data isaccumulated or a display of each iteration provided at the option of theoperator. The operator further has the option to bypass setting thecytoplasm boundary and DNA boundary once they have first been set for aspecimen image.

The system program for DNA quantitation is, in general, a menu drivenprogram which allows the operator to interactively communicate with theimage analysis system 13 to produce the quantitation of nuclear DNA byimage analysis. The system program displays a plurality of images orinstruction screens on the instruction monitor 62 which include menusfrom which to select the various functions needed for performing aquantitative nuclear DNA assay. FIG. 17 illustrates the screenarchitecture of the system and the paths that the system takes betweenscreens. Examples of two of the system screens, the calibrate screen A14and the analysis screen A16, which appear on the instruction monitor 62are pictorially illustrated in FIGS. 15 and 16, respectively.

Returning to the reference numerals in FIG. 17, the system program maybe run by calling it as an application program of the operating systemA10. Selection of the system program by the operating system A10produces the main screen A12 on the monitor 62. From the main screen A12the operator can select a calibrate screen A14, an analysis screen A16,or exit back to the operating system A10. While displaying the calibratescreen A14, the instrument can be calibrated to provide the backgroundor reference light settings which will be used in the measurement of theassay. Once the light calibration is complete the operator can selectthe analysis screen A16 which is used to measure and classify the cellobjects of the assay technique.

One of the options in analysis screen A16 is to adjust the blue boundarywhich assists in forming the nuclear areas. Another of the options is toadjust the red boundary which produces the adjust red boundary screenA20. Once the nuclear and cytoplasmic areas have been bounded by thescreens A18, A20, the operator can select the analysis screen A16 toactually do cell measurement, classification and to generate reports.Exits from the adjust blue boundary screen A18 and from the adjust redboundary screen A20 are to the analysis screen A16, which can then exitback to the main screen A12.

In this manner an advantageous screen architecture is formed which canbe easily used and understood by the operator. This screen structurefacilitates the interactive measurement of the nuclear DNA of theparticular cell subpopulation under study. The instruction screensprovide an interactive use of the digital imaging system which combinesthe power of the system software and hardware with the judgment andknowledge of the operator. The screen structure automates the assay taskof nuclear DNA quantitation while still permitting the operator toselectively choose the input data and control the process to aconsiderable degree.

Each screen A12-A20 contains a menu of the functions permitted for usewhile that particular screen is being displayed on the instructionmonitor 62. The function that the system is to currently execute in aparticular menu is chosen by the operator with a cursor movement methodusing the standard cursor control keys of keyboard 36. While aparticular screen is being shown on the monitor, the cursor movementkeys are operable to position the cursor next to a particular functionlisted on the menu of that screen. While the cursor highlights thefunction by its position, the operator may select the function forexecution by pressing the enter key.

The main screen 12 displays the main menu A22 illustrated in FIG. 18.The main menu A22 provides five choices which include 1) a specimenfunction A24, 2) a label function A26, 3) a set light function A30, 4) acalibrate function A32, and 5) an exit function A28.

The calibrate screen A14 displays the calibrate menu A34 illustrated inFIG. 19. The calibrate menu A34 provides seven choices which include 1)a check light function A36, 2) a set XY function A38, 3) a focusfunction A40, 4) a measure function A42, 5) an XY-1 function A44, 6) ananalyze function A46, and 7) a main function A48.

The adjust blue boundary screen A18 displays the adjust blue boundarymenu A50 illustrated in FIG. 20. The adjust blue boundary menu A50provides five choices which include 1) a set step size-B function A52,2) a toggle-B function A54, 3) an increase-B function A56, 4) adecrease-B function A58, and 5) an exit function A60.

The adjust red boundary screen A20 displays the adjust red boundary menuA62 illustrated in FIG. 21. The adjust red boundary menu A62 providesfive choices which include 1) a set step size-R function A64, 2) atoggle-R function A68, 3) an increase-R function A70, 4) a decrease-Rfunction A66, and 5) an exit function A72.

The analysis screen A16 displays the analysis menu A74 illustrated inFIG. 22. The analysis menu A74 provides sixteen choices which include 1)a del-type function A76, 2) a classify function A78, 3) afocus-2function A80, 4) a CK-light function A82, 5) a select 2ndfunction A84, 6) an area 1-2 function A86, 7) a display XY function A88,8) a clear function A90, 9) a XY-2 function A92, 10) a report functionA94, 11) a scale function A96, 12) a boundary-B function A98, 13) aboundary-R function A100, 14) a main function A102, 15) a mask functionA104, and 16) a disappear function A106.

The label function allows a user to enter information regarding patientidentification, accession number, and DNA conversion number. The DNAconversion number is the number that the first and second peak massesare divided by to get the first and second peak indexes. Initially, thenumber is set to a standard 7.18 picograms/cell for normal human cells.However, the apparatus may be used to measure non-human cells and theindex may be changed to that desired. The DNA index number must begreater than or equal to 1.0 and less than or equal to 99.99. If theconversion number is not within that range, the user is not allowed toselect the analyze option in either the main or the calibration screens.

The three lines of information entered during the label function willappear on every screen except the X, Y field coordinate screen. Thelabel operation is exited by pressing either the enter or escape key.Pressing the enter key will save any changes that were made to the threelines of information. Pressing the escape key will ignore any changesthat were made to the three lines. The information stored during thelabel function will not be saved when the program is exited.

Selecting the calibrate function will cause a change of the display oninstruction monitor 62 from the main screen to the calibration screenillustrated in FIG. 15. The calibration screen whose options are shownin FIG. 19 are those necessary to perform calibration of the instrumentfor optical density and for staining factor on the control cells. Acalibration of the apparatus 11 is to be performed every time a newslide is selected to normalize the light level and staining factor.

Selecting the analyze function will cause a change of the display fromthe main screen on monitor 62 to the analysis screen as illustrated inFIG. 16. The analysis screen contains the menu for the functions thatare necessary to perform data acquisition and DNA measurements on thespecimen cells. These functions are more fully set forth in FIG. 22.Three criteria must be met in order to select the analyze function.First, the set light function in the calibration screen must have beensuccessfully performed at least once. The set light function issuccessful when the current image is blank and the light level isbetween 129 and 131. Secondly, the calibration control cell count mustbe between 50 and 512. Finally, the DNA conversion number must begreater than or equal to 1.0 and less than or equal to 99.99.

The exit function allows the user to terminate the operation of theprogram from the main screen. Pressing the escape key is the same asselecting the exit function. When the exit operation is specified,either by selecting exit or pressing escape, the user will be asked toconfirm his command to exit. To accept the confirmation, the userselects the yes key. To reject the confirmation, he selects the no keyor presses the escape key.

The options of the calibration menu will now be more fully explained.The set X, Y of function A38 provides the setting of the origin for theslide X, Y coordinate system. This function sets the current image orfield location as the origin by zeroing a pair of location registers inthe software. Generally, the microscope platform 51 is moved until aeasily recognized landmark is visible, such as cross 53. This landmarkis then used to rezero the coordinate system to provide a means ofrelocating previously measured fields. The set X, Y function is usedevery time a new slide is selected. If the set X, Y function has notbeen executed, then the X, Y functions of the calibration and analysisscreens and the functions in the X, Y field coordinates screen will notwork properly. The set X, Y function can only be used when thecalibration control cell count is equal to zero. If the microscopeplatform 51 is being moved when the set X, Y operation is in execution,then the coordinate origin will be in error. The program provides amessage on the screen to notify the operator when the set X, Y operationis successful. In response to the function not being successful, theoperator merely reselects set X, Y from the menu and attempts thefunction again.

The measure function A42 is used to perform the control cell or controlobject calibration for normalizing the staining factor. When the measurefunction is selected, the camera image acquisition will stop and thecursor 170 on the calibration screen will move to the words "measureoperations" in FIG. 15. When the cursor 170 is at this location, theuser can specify measure operations by activating the numeric lock key.An identifier such as a magenta colored box will be placed around anidentified cell object. By using a number of key operations, theoperator can perform an interactive selection and rejection processwhich will be more fully explained hereinafter.

During control cell calibration, the operator moves the microscope stageby turning the conventional X and Y knobs 12 and 17 (FIG. 1) to shiftthe control cell objects 40 into view on the image monitor 37. When anindividual cell object 40 is within a box or identifier border 75, theoperator presses a key on the keyboard 36 to enter measurement of thesummed optical density for that control cell object. After a suitablenumber of control cell objects have been analyzed, the operator will beprovided with a histogram such as shown in FIG. 15 on the instructionmonitor 62 which shows the operator the control cell object ploidydistribution as having a relative quantity of DNA. Internally within thesystem control 22, the summed relative optical density values actuallymeasured for the control cell objects are compared to a predeterminedstandard or reference amount of DNA which the control cells are known tohave. The actual summed optical density found by the operator is dividedinto the stored reference DNA value to provide a factor by which toadjust the extinction coefficient for deviations in the stain from aperfect staining.

The XY-1 function A44 when selected displays on the calibration screenthe X, Y coordinates of the current image or field, on image monitor 37.The coordinates will be continuously displayed until the user presses akey (except CTRL, ALT, or SHFT). Thus, if the same origin for the slide14 was set, the operator can, by positioning the platform 51 andwatching the coordinates change, find the same image which waspreviously recorded. The set X, Y function A36 must have beensuccessfully performed previously in order for the X, Y function to beselected.

The FOCUS-1 function A40 provides color enhancement to the image so thatthe user can perform more precise focusing of the image. The systemcontrol 22 automatically provides different colors for gradations ingrey level in the image. The operator then adjusts the focusing means ofthe microscope 15 until the object being focused on, for example an edgeof the border 54, shows a clear color demarkation. This is an indicationthat the two separate levels or grey scale of the edge are in focus.This is much more difficult without color because the two grey levelsmay be close together and undiscernible without the color enhancement.The set light function A30 must have been successfully performed atleast once in order to select the focus function. To restore the imageto its original color, the focus function is selected a second time. Ifthe color enhanced image is present when the user selects the measurefunction, the image will automatically be returned to its originalcolor.

Selecting the analyze function will change the display from thecalibration screen to the analysis screen. The analysis screen providesa menu of functions shown in FIG. 22 which are necessary to perform theDNA measurements on the cellular material. Three criteria must be met inorder to select the analyzed function. First, the set light function inthe calibration screen must have been successfully performed at leastonce. Secondly, the calibration control cell count must be between 20and 512. The analyze function in the calibration menu works the same wayas the analyze function in the main menul.

The analyze function options in the analysis menu are more fully shownin FIG. 22.

The check light-2 function A82 calculates the light level of the currentimage. The light level value is displayed on the analysis screen by thewords "light level" in FIG. 16.

The select-2nd function A84 allows the user to select the second peak onthe histogram displayed on the analysis screen. The mass, DNA index, andthe area of the second peak are displayed on the screen under the words"second peak." The select-2nd function cannot be selected when the showncell count is equal to zero. The shown cell count is displayed by theword "shown." After the select-2nd function has been selected, thecursor will move to a set of arrows and the current second peak locationon the histogram will be highlighted in yellow. Initially the right mosthistogram data location is chosen as the second peak. Selecting the leftarrow moves the second peak location to the left and the user selectsthe right arrow to move the second peak location to the right. Everytime an arrow is selected, the current peak data on the screen will beupdated.

Below the histograms horizontal axis, one of three symbols will appearunderneath the second peak location. A "less than" symbol will appear ifthe second peak lies in area one. A "greater than" symbol will appear ifthe second peak lies in area two. An up arrow symbol will appear if thesecond peak lies in neither area one nor area two. The reason for thethree symbols is so that the second peak location can be identifiedafter the select-2nd operation is exited. The vertical yellow linedisappears once the select-2nd function is exited. The users presses theESC key to exit the select second operation. The second peak data willalso be automatically cleared when one of the following analysis screenfunctions is selected: clear, report, scale, or main.

The classify function A78 allows the user to classify the cells orobjects in the current image. After the classify function has beenselected, the user will be asked to confirm the operation. To accept theconfirmation, the user will select the yes key, and to reject theconfirmation, the user will select the no key or press the ESC key. Ifthe classification function is confirmed, camera acquisition stops andthe cursor will move by the words "classify operation". When the cursoris at this location, the user can specify the classify operations byactivating the numeric lock to enable these functions. As was the casein the measure function, a magenta colored box will be placed around acurrent cell and the operations allow the user to move this cellidentifier through the image to identify and classify the cells therein.

The display X, Y function A88 will change the display from the analysisscreen to the X, Y field coordinates screen. The X, Y field coordinatescreen will display the X, Y coordinates of the first 512 images thathave been classified and stored. Also, the screen contains the functionsthat allow the sorting of the image fields by coordinates. The set X, Yfunction in the calibration screen must have been successfully performedbefore the display X, Y function is selected.

The X, Y field coordinate screen has several functions. One of thefunctions, "nearest" sorts the X, Y coordinates according to thedistance from the current X, Y field position. The X function will sortthe X, Y coordinates according to the X coordinate value. If there is atie, then the Y coordinate value will determine the sort order.Similarly the Y function will sort the X, Y coordinates according to theY coordinate value. If there is a tie, then X coordinate value willdetermine the sort order. The "field#" function will sort the X, Ycoordinates according to the coordinates field number. The field numberis the order in which the images were classified.

The page up function allows the user to display the previous page of X,Y coordinates, if any, and the page down function allows the user todisplay the next page of X, Y coordinates, if any. The exit functionchanges the display from the X, Y field coordinate screen to theanalysis screen. Pressing the escape key is the same as selecting theexit function.

Selecting the X, Y function displays the X, Y coordinates of the currentfield. The coordinates will be continuously displayed until the userpresses a key (except CTRL, ALT, and shift). The set X, Y function inthe calibration screen must have successfully been performed before theX, Y function is selected. The X, Y function in the X, Y fieldcoordinate screen works the same way as the X, Y function in thecalibration and analysis screens.

The clear-2 function A90 will clear all analysis related areas of data.After the clear function has been selected, the user will be asked toconfirm the clear operation. To accept the confirmation, the userselects the yes key, or to reject the confirmation, the user will selectthe no key or press the ESC key.

The focus-2 function A80 provides color enhancement to the image so thatthe user can perform more precise focusing of the image. The focus-2function in the analysis screen works the same way as the focus functionin the calibration screen previously described.

The area 1-2 function A86 allows the user to specify two areas in thehistogram displayed in the analysis screen. The purpose of this functionis to identify the cell counts in certain areas in the histogram. Thearea 1-2 function cannot be selected when the shown cell count is equalto zero. The cell counts are displayed at the lower right portion of thescreen. After area 1-2 is selected, the cursor will move to a row ofnumbers that is below the histogram horizontal axis. The row of numbersallows the user to specify the locations of area 1 and area 2. The usertypes a "1" to specify that the current histogram position belongs toarea 1. The user types a "2" to specify that the position belongs toarea 2. The user types a "0" to specify the current histogram positionbelongs to neither area 1 or 2. The user is allowed to specify an area 1without an area 2, but cannot specify an area 2 without an area 1. Whenboth areas are specified, area 1 must be specified to the left of area2. The area must be specified as continuous. To exit the area 1-2function, the user presses the enter or ESC keys. If the user pressesthe enter key, area 1 of the histogram will be highlighted in green andthe area 2 will be highlighted in magenta. The area cell counts willalso be displayed. Pressing the ESC key will cause the program todisregard any of the changes that were made. Area 1 and area 2 data willautomatically be cleared when one of the following functions isselected: classify, clear, reports, scale or main.

The analysis function is more fully described with respect to FIGS. 13and 14. The operator will select a number of field locations 360, 361,and 362 in the slide specimen area 58 for analysis. The operator willadjust the X and Y knobs 12 and 17 for the microscope stage 51 to movethese fields into view on the image monitor 37 a first field of specimencell objects to be analyzed for DNA content as well as for cellmorphology if desired (FIG. 13). The program will place a box, forexample at 300, over a particular specimen cell object being displayedon the monitor 37 and then the operator will use a key to cause thescanning of the pixels (picture elements) of the specimen object toclassify the cell in a manner similar to that disclosed in U.S. Pat. No.4,453,266 to give summed optical density for the cell specimen objecti.e., a stained cell nucleus, as well as its area, its roundness, andother classification information.

Also, the operator has on the keyboard 36 several cell classificationkeys to be manually operated and the operator depresses one of the keysof a known category such as a type 0 normal cell; a type 1 cancer cell;a type 2 cancer cell; a type 3 cancer cell; and etc. On the monitoringscreen 62 there will be an analysis histogram displaying the DNA contentof the cells in the field. The operator selects a number of cells ineach field or area and then moves the microscope stage to position anumber of different fields of specimen cells into view and takes andanalyzes a number of these specimen cells until the operator feels hehas enough cells for a representative sample.

A histogram, will at this time be displayed on the instruction monitorscreen 62 which shows the number of cells of a particular DNA contentand shows the DNA content averages for each of the reference peaks, suchas shown in FIG. 16. By depressing a print key, on the keyboard 36 theoperator may print out the histogram shown in FIG. 16 on the printer 38.The data for the specimen cells is also stored internally within thesystem control 22 for later recall and comparison with data of any newspecimen from the same patient for analysis relating to the progress orregression of a patient.

The operation of the manual classification for the analysis functionwill be now more fully described with respect to FIG. 13 where there isshown a visual field which has been previously stored in the instrument.The field contains a number of cell objects which are to be classifiedand measured as to DNA content. When the program initially comes intothis mode of operation, the first object in the field will be identifiedby scanning the pixels of the field in a raster like manner until a cellobject is recognized. Once a cell object is recognized, anidentification means such as box 300 is drawn around the object. Thisprovides a visual identifier for the operator to determine which cellobject is presently being measured. In addition to the measurement, theoperator is provided with a number of options from the analysis menu.The primary option that an operator has is to classify a current objectin block A78. He accomplishes this by pressing one of the numeric keys 0through 5 which automatically puts the cell object of the identifyingbox 300 into the classification selected. If the object identified isdebris, not an abnormal cell, or not an identifiable cell object, theoperator can reject the current object by selecting a 9 on the keyboardas indicated. After the classification or rejection of the object in box300, the operator can move the identification box to the next unmeasuredobject. An operator accomplishes this by pressing the keys CTRL/F2 whichcauses the program to erase the box 300 and search for the nextidentifiable cell object. This cell object is found, and then anotheridentifying box 302 is drawn around it to indicate to the operator thefunction has been accomplished. In this manner, the entire group of cellobjects can be classified and measured or rejected by repeating thisprocess. In this manner, the program steps through the analysisprocedure from object 300 to 302, 304, 306, 308, 310, etc.

Further, if one of the cell objects to be classified does not look likethe operator thinks it should and, it cannot be put in one of theprevious classifications, or for some other reason the operator believeshe has classified a previous object by mistake, then by pressingCTRL/F1, he can move the identifying box back to the previously measuredobject. After identifying all cell objects in the particular field beingdisplayed, the operator has the option of going to another field bymanipulating the X, Y positioning mechanism to provide more cells forthe particular analysis.

When the operator has determined that enough cell objects had beenanalyzed, he may either terminate the analysis function by pressingeither the enter key or escape key. If he terminates the analysisfunction by pressing the enter key, then the data assembled from each ofthe measurements will be saved. However, if the analysis function isterminated by pressing the ESC key, then the data will not be saved.

The report function A94 allows the user to specify which cellclassifications are to be included in the histogram shown on the screenof the instruction monitor 62. After the report function has beenselected, the cursor can be moved to an option list which will allow theoperator to specify the cell types. The following table specifies whichkey the operator presses in order to select a particular cell type.

    ______________________________________                                               CELL TYPE      KEY                                                     ______________________________________                                               normal         0 or n                                                         1              1                                                              2              2                                                              3              3                                                              4              4                                                              lymphocyte     5 or L                                                  ______________________________________                                    

Any combination of the types for the report data is allowed. The programwill ignore any other characters than those listed in the table. Theoperator exits the report operation by pressing the enter or escape key.If the operator presses the enter key, he will change the types of cellsin the histogram to those which were specified. However, if the escapekey is selected, the program will ignore any changes that were made andreturn normally. The function area 1-2 data and the second peak datawill automatically be cleared when the report operation is performed.

The scale function A96 allows the operator to change the scale of thehorizontal axis of the histogram provided on the analysis screen. Thereare three scales to choose from, 0-16, 0-32, and 0-64. If the scalefunction is selected when the current scale is 0-16, then the new scalewill be 0-32. If the scale function is selected when the current scaleis 0-32, then the new scale will be 0-64. Likewise, if the scalefunction is selected when the current scale is 0-64, then the new scalewill be 0-16. In this function the area 1, area 2, and second peak datawill automatically be cleared when the scale operation is performed.

The boundary functions A98, A100 will change the display on instructionmonitor 62 from the analysis screen to the respective adjust boundaryscreen. The adjust boundary screen contains functions that are necessaryto change the cell boundary, i.e., threshold. While addressing theboundary screen, camera image acquisition will be halted.

The set step size function of FIGS. 20 and 21 allows the operator tochange the amount by which the boundary will change when one of thearrow keys is selected. The value must be in the range of 0-129. Afterthe step size is selected, the cursor will move to the location on thescreen where the user can type in a new step size value. To exit thestep size function, the enter or escape keys are used. Pressing theenter key will save the step size change where pressing the escape keywill ignore any change that was made. Initially, the step size value isequal to one.

The increase function A56, A70 will increase the cell boundary by thevalue of the step size and the decrease function A58, A66 will decreasethe cell boundary by the value of the step size. The exit functions A60,A72 change the display from the adjust boundary screens back to theanalysis screen. Pressing the escape key is the same as selecting theexit function.

In general, an interactive data collection and analysis scheme is usedby the apparatus for the collection of specific parameters for both thecalibration cell objects and the specimen cell objects. Each field whichis selected is displayed on the image monitor 37 and either the measureoperation of the calibration screen or the classify operation of theanalysis screen is chosen.

A software flow chart of an subroutine providing the interactiveoperations for the calibrate key operations and the analysis keyoperations, FIGS. 15 and 6 and is illustrated in the referenced Bacusapplication. When the operator selects either the measure operations orthe classify operations, this program is called to generate theselection process for both the calibration cell objects and the specimencell objects. The program begins by performing a raster scan of thestored image pixel by pixel until it finds a pixel greater than thethreshold value. If no pixel is found which is greater than thethreshold, a determination of whether the scan is complete is made. Ifnot, the scan is continued until all pixels in the image field aretested. After all pixels have been tested, the scan parameters are resetand the cell object array updated.

At the time an image pixel is determined to be greater than thethreshold, the program will label the object. The operation of labellingwill now be more particularly described. The individualized cell objectsin the digitized image are located by a scene analysis technique inwhich the raster scan is made of the digitized image to locate any pixelabove the critical threshold. The technique then performs a fourneighbor analysis of adjacent pixel elements and continues in arecursive manner locating "neighbors of the neighbors" which are abovethe threshold until the entire region of a cell object is defined. Thistechnique is preferred to other scene analysis techniques, such as localboundary found from a gradient image, because it is fool proof indistinguishing the true region of a cell, particularly those cellshaving irregular or spiculed projections.

The four pixels (top, bottom, right side, and left side) surrounding theinitially located pixel which are contiguous therewith are examinedsequentially to identify the next pixel with a optical density or graylevel value above the threshold. For instance, if the pixel locatedabove the first pixel is not above the threshold, it is discarded fromthe labelling routine. The next pixel (right side) in a clockwisedirection is then examined and may be above the threshold. If so, thatpixel is then identified and stored in memory with the pixel as being aportion of the region of a cell. Next the address and density of thepixel found is stored in a pushdown list and the four neighboring pixelsof that pixel are examined in the same clockwise order. This continuesin a recursive manner until no neighbors are found above a threshold fora particular pixel. At this point the prior pixels on the pushdown listare reexamined to continue the neighbor search process until the entirenumber of pixels defining a region, i.e., the cell object has beenidentified. Thus, each of the pixels above the threshold of the regionare identified and a complete enclosed region has been defined for acell.

Once a cell object is labelled, a cell object table is set up for theobject. The table lists the address of its entry point pixel, the numberof pixels in the object, the X, Y points for the minimum and maximumpoints of the object, a count of the pixels in the perimeter of theobject, the sum of the optical density of the object pixels, anyclassification provided for the object, and the X, Y coordinates of thefield to which the object belongs. A plurality of the cell object tablescomprise a temporary array, called a field array, which is used to storethe interactive data developed for the present field image underconsideration.

Next, a box or identifying border is placed around the object using theX, Y limits. This mode identifies a particular object in the field forthe operator. A key handler is entered to obtain a key press from theoperator to determine which of the key functions of the classificationfunction are to be accomplished. The key handler further determineswhich operation, either for calibration or analysis, is to be performedand only those keys which are associated with the present mode areenabled, all others are locked out. Once a key has been obtained, theprogram will determine which function was selected and the progress ofthe routine.

Keys 0-5 as detected provide for the acceptance of a calibration objector the classification of a specimen object. If such key is detected,then the object is colored (red) to indicate to the operator that it hasbeen accepted or classified. The operator classifies the cell objectsinto different categories based upon visual clues such as morphology andthe optical markings. The cells for analysis can be classified into anormal class 0, or one of several abnormal classes 1-5. The data classof the object is stored in its place in the associated object datatable. Calibration objects are classified as type 0 or normal. Theprogram then returns to where the image scan registers are incrementedto scan the field for the next object.

Alternatively, if the key press was a 9 this means either a calibrationcell object was rejected or a current specimen cell object was rejected.Thus, the rejected cell object is colored in a different color (white)than an accepted or classified cell object, and the program returns tothe scanning routine to find another object. Coloring the cell objectalerts the operator that the object has been analyzed in this field,coloring the object another color differentiates the object from anaccepted or classified cell objects.

If, however, the key press is a CTRL/F1, then the operator desires tomove the identifying box to the last previously measured object. Theprogram will then interrogate the field array to find the last objectpointer. This pointer is used to create the box around the previous cellobject before getting another key press. By using a series of CTRL/F1keys the operator may selectively move the identifying box thepreviously measured cell object the previously measured cell object in areverse direction. If, after the box is placed around a particular cellobject, the operator desires to reclassify that cell object, he then hasthe option of classifying it with the keys 0-5 in block A326.

The identifying box may be moved to the next unmeasured cell object byselecting the key CTRL/F2. The key, if found, immediately returns theprogram control to the image scan entry. The effect of this operation isto allow the operator to skip the present cell object and move theidentifying box to the next cell object without either rejecting oraccepting the present cell. A series of CTRL/F2 presses will move thebox forward through the cells without measuring them.

If all of the cell objects in a particular field appear normal asspecimen cells, or as is generally the case with control cells they areacceptable, the operator may want to classify them all automatically. Toaccomplish this, an operator presses the key CTRL/F3. This key press isdetected and transfers control to where the automatic mode flag is set.The program then returns to the entry of the image scan. However,instead of going through the normal sequence of placing a box around thenext object and waiting for a key press, the program will loop toautomatically classify the rest of the cells of a field.

Another option that the operator can select is the cell cutting functionwhich is entered by pressing the key CTRL/F4. This key is detected andtransfers control to the cell cutting function operation. When the CTRLand F4 keys are pressed, the user enters the cell cutting mode. While inthis mode, the user is permitted to make cut lines inside the identifierbox. The operator cannot make a cut line over a pixel that belongs to ameasured or a rejected cell. A measured cell is a cell that has beenclassified as type 0, 1, 2, 3, 4, or 5. Numeric lock must be activatedin order to perform a cell cutting operation. A cross hair is locatedwhere the cut is to take place. The following table lists the cellcutting operations that can be performed plus the key that must bepressed in order to select the desired operation. The function allowsthe splitting of overlapping cells by artificially making a perimeterbetween two areas, a cut. Thus, the labelling routine will only labelone area as a cell object.

    ______________________________________                                        (KEY)         (ACTION)                                                        ______________________________________                                        0             Turn splitting on and off                                       1             Go down and left one step                                       2             Go down one step                                                3             Go down and right one step                                      4             Go left one step                                                5             Go to the center of the box                                     6             Go right one step                                               7             Go up and left one step                                         8             Go up one step                                                  9             Go up and right one step                                        ENTER         Re-do last step (up to 100 pixels)                              ESC           Exit cell splitting mode                                        ______________________________________                                    

A step is three pixels. When beginning a new cut, the first pixel willnot be cut. For operation 5, the cross hair will not move if the centerpixel belongs to a measured or rejected cell.

After the cell cutting is performed, the scanner registers are set tothe entry point of the particular object cut. The program then returnsto the scan entry. Because the cell object has the same entry point buta different perimeter, the labelling routine will label the cell objectas now cut.

Another option that the operator has is the ability to select any objectwithin a field. The selection of this mode is accomplished by pressingthe CTRL/F5 key.

When the CTRL and F5 keys are pressed, the user enters the selectionmode. Numeric lock must be activated in order to perform a selectionoperation. A cross hair will appear at the current selection point. Thefollowing table lists the selection operations that can be performedplus the key that must be pressed in order to select the desiredoperation.

    ______________________________________                                        (KEY)       (ACTION)                                                          ______________________________________                                        0           Select cross hair movement step size                                          [5 or 15]                                                         1           Go down and left one step                                         2           Go down one step                                                  3           Go down and right one step                                        4           Go left one step                                                  5           Go to the center of the image                                     6           Go right one step                                                 7           Go up and left one step                                           8           Go up one step                                                    9           Go up and right one step                                          ESC         Exit selection mode                                               ______________________________________                                    

When the selection mode is exited, the box will move to the firstunmeasured cell after the selection point. If there are no cells afterthe cross hair, the box will go to the next unclassified cell.

After the object is selected by the above described technique, thescanner registers are set to the entry point of that particular objectin block A348 and the program returns to the scan entry in block A300.This creates the identifier box around the object using its X, Y limitsand provides the operator with the option of then pressing another keyand performing other measurements and classifications on that selectedobject.

Another function is provided by key CTRL/F6. This feature provides anoperator with the ability to move the identifier box forward by readingthe next cell object pointed to and then drawing around the box thechosen object in block A313. The keys CTRL/F1, CTRL/F2 thereby allow anoperator to quickly revise previous cell classification by steppingforward and backwards, respectively, through the pointers of thepreviously measured cells.

When the enter key is sensed, the cell object array is updated with thepresent field array to store all of the data collected for theparticular objects in the field. Alternatively, the sensing of theescape key returns the program immediately to the place in the softwarewhere it was called.

It will be appreciated that the illustrated control 22 has beenprogrammed to do the cell classification and optical density analysis.Such classification and analysis is similar to that outlined in U.S.Pat. No. 4,453,266 for the classification of red blood cells and thepresent invention can be particularly useful in the analysis of redblood cells wherein the optical density of the hemoglobin content ismeasured rather than the DNA content as above described. As common inred blood cell analysis, the red blood cells need not be stained forimage enhancement so that the staining calibration step may beeliminated for red blood cells when using the specific wave length oflight specified in the aforementioned Bacus patents.

A further use of the present invention is to provide a precisemeasurement of hemoglobin in actual picograms for calibrating otherinstruments such as a Coulter counter. In such a process, the controlblood cells 40 will have a known predetermined hemoglobin value and thespecimen blood cells 52 of unknown hemoglobin value will be placed onthe specimen area 58. Then the apparatus will be calibrated to show thehistogram for the hemoglobin content of the specimen cells 52.

It will also be appreciated that the various calibration steps may beeliminated or combined and done simultaneously rather than done in theorder and in the sequence and in the manner described for the preferredembodiment of the invention in making a DNA analysis.

While a preferred embodiment of the invention has been illustrated, itwill be obvious to those skilled in the art that various modificationsand changes may be made thereto without departing from the spirit andscope of the invention as defined in the appended claims.

What is claimed is:
 1. A method of marking selected cells in asubpopulation and for measuring the DNA content of predetermined classesof cells in the subpopulation by image analysis means, said methodcomprising the steps of:first, staining the cells in the subpopulationwith an alkaline phosphatase technique using a chromogen substrate offast red without harming the staining capabilities of DNA in the nucleusof said cells which includes the steps of, reacting a cellularcytoplasmic or membrane protein with a monoclonal antibody specific tosaid protein in at least one of the cell cytoplasm or the cell membraneof said cells, optically enhancing said monoclonal antibodies bound tosaid protein with said alkaline phosphatase technique using a chromogensubstrate of fast red thereby marking said cells which include saidprotein as to antigenic type, second, staining DNA in the nucleus in allthe cells in the subpopulation with a Feulgen technique without harmingthe staining capabilities of the chromogen substrate, and third,measuring the DNA content of the marked cells by said image analysismeans.
 2. A method of marking selected cells in a subpopulation as setforth in claim 1 wherein said protein is selected from the groupconsisting of cytokeratin and leukocyte common antigen.
 3. A method ofmarking selected cells in a subpopulation as set forth in claim 1wherein said step of staining the DNA includes:staining the DNA of thecells with Thionin.